Deblur Rapidly Resolves Single-Nucleotide Community Sequence Patterns

Almasi‐Hashiani, Amir; McDonald, Daniel; Navas-Molina, José A.; Kopylova, Evguenia; Morton, James T.; Xu, Zhenjiang Zech; Kightley, Eric P.; Thompson, Luke R.; Hyde, Embriette R.; González, Antonio; Knight, Rob

doi:10.1128/msystems.00191-16

Cited by 1,500 publications

(1,204 citation statements)

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“…2b). We therefore employed a reference-free method, Deblur 21 , to remove suspected error sequences and provide single-nucleotide resolution ‘sub-OTUs’, also known as ‘amplicon sequence variants’ 22 , here called ‘tag sequences’ or simply ‘sequences’. Because Deblur tag sequences for a given meta-analysis must be the same length in each sample, and some of the EMP studies have read lengths of 90 bp, we trimmed all sequences to 90 bp for this meta-analysis.…”

Section: A Standardized and Scalable Approachmentioning

confidence: 99%

A communal catalogue reveals Earth’s multiscale microbial diversity

Thompson¹,

Sanders²,

McDonald³

et al. 2017

Nature

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2,095

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A primary aim of microbial ecology is to determine patterns and drivers of community distribution, interaction, and assembly amidst complexity and uncertainty. Microbial community composition has been shown to change across gradients of environment, geographic distance, salinity, temperature, oxygen, nutrients, pH, day length, and biotic factors 1-6 . These patterns have been identified mostly by focusing on one sample type and region at a time, with insights extra polated across environments and geography to produce generalized principles. To assess how microbes are distributed across environments globally-or whether microbial community dynamics follow funda mental ecological 'laws' at a planetary scale-requires either a massive monolithic cross environment survey or a practical methodology for coordinating many independent surveys. New studies of microbial environments are rapidly accumulating; however, our ability to extract meaningful information from across datasets is outstripped by the rate of data generation. Previous meta analyses have suggested robust gen eral trends in community composition, including the importance of salinity 1 and animal association 2 . These findings, although derived from relatively small and uncontrolled sample sets, support the util ity of meta analysis to reveal basic patterns of microbial diversity and suggest that a scalable and accessible analytical framework is needed.The Earth Microbiome Project (EMP, http://www.earthmicrobiome. org) was founded in 2010 to sample the Earth's microbial communities at an unprecedented scale in order to advance our understanding of the organizing biogeographic principles that govern microbial commu nity structure 7,8 . We recognized that open and collaborative science, including scientific crowdsourcing and standardized methods 8 , would help to reduce technical variation among individual studies, which can overwhelm biological variation and make general trends difficult to detect 9 . Comprising around 100 studies, over half of which have yielded peer reviewed publications (Supplementary Table 1), the EMP has now dwarfed by 100 fold the sampling and sequencing depth of earlier meta analysis efforts 1,2 ; concurrently, powerful analysis tools have been developed, opening a new and larger window into the distri bution of microbial diversity on Earth. In establishing a scalable frame work to catalogue microbiota globally, we provide both a resource for the exploration of myriad questions and a starting point for the guided acquisition of new data to answer them. As an example of using this Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of r...

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Section: A Standardized and Scalable Approachmentioning

confidence: 99%

A communal catalogue reveals Earth’s multiscale microbial diversity

Thompson¹,

Sanders²,

McDonald³

et al. 2017

Nature

Self Cite

2,095

1,758

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“…The pooled library was sequenced on the Illumina MiSeq sequencing platform with a MiSeq Reagent Kit v2 and paired-end 150 cycles. All data were processed and analyzed using the QIIME2 software suite [8] and Deblur [9]. Counterintuitively, single PCR reactions yielded significantly more reads than triplicate PCR reactions (mean ± SEM: 10,821 ± 298 versus 10,029 ± 262, respectively, paired T-test p = 0.0003), and fewer dropouts (Figure 1A).…”

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confidence: 99%

Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

et al. 2019

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Conventional wisdom holds that PCR amplification for sequencing should employ pooled replicate reactions to reduce bias due to jackpot effects and chimera formation. However, modern amplicon data analysis employs methods that may be less sensitive to such artifacts. Here we directly compare results from single versus triplicate reactions for 16S amplicon sequencing and find no significant impact of adopting a less labor-intensive single-reaction protocol.

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“…DNA was prepared for 16S rRNA gene amplicon sequencing as previously described (7). The demultiplexed fasta files were obtained from qiita.ucsd.edu (study ID 10178), and operational taxonomic units (OTUs) were detected using Deblur (8). Raw reads were submitted to the European Bioinformatics Institute under accession no.…”

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confidence: 99%

DNA Extraction for Streamlined Metagenomics of Diverse Environmental Samples

et al. 2017

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A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. The data demonstrate that any effect of extraction method on sequencing results was small compared with the variability across samples; however, the KingFisher platform produced the largest number of high-quality reads in the shortest amount of time. Based on these results, we have identified an extraction pipeline that dramatically reduces sample processing time without sacrificing bacterial taxonomic or abundance information.

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Deblur Rapidly Resolves Single-Nucleotide Community Sequence Patterns

Cited by 1,500 publications

References 21 publications

A communal catalogue reveals Earth’s multiscale microbial diversity

A communal catalogue reveals Earth’s multiscale microbial diversity

Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

DNA Extraction for Streamlined Metagenomics of Diverse Environmental Samples

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